In the prior funding period we obtained results strongly suggesting that oscillatory fluid flow due to loading is an important cellular physical signal for both osteoblasts and osteocytes. Utilizing our custom built dynamic flow system, we were able to show that oscillatory fluid flow can regulate cell metabolism via intracellular calcium mobilization, prostaglandin E2 release, and MAP kinase activity in the absence of other physical or biochemical signals. However, we have not uncovered the molecular mechanotransduction mechanism activated by oscillatory fluid flow. Candidates can be expected to experience load due to flow and have biochemical signaling potential. This conceptual model is supported by our observation made in the prior funding period that degradation of membrane proteoglycans extending into the flow field has a dramatic effect on the response to flow. Also, we have preliminary indications that actin and focal adhesion kinase (FAK) are involved fluid flow induced signaling. Thus, the central hypothesis of this five year project is that oscillatory fluid flow regulates bone cell metabolism via a molecular mechanism involving forces experienced by the cytoskeleton and transmitted through focal adhesion sites to integrins. To test this hypothesis we will undertake a systematic multilevel evaluation of cell structural proteins to include actin, integrins, and linker proteins both in terms of the effect of oscillatory flow on these proteins (aim 1) and the role of each in transducing the response to flow (aim 2). Additionally, strong evidence from our laboratory and others suggests specific involvement of focal adhesion kinase (FAK) tyrosine phosphorylation. This combined with recently developed molecular tools targeting FAK phosphorylation motivate us to perform a more in-depth investigation of two specific FAK signal pathways (aim 3). Finally, utilizing a novel microfabricated flow chamber, we will determine if the osteocyte process is a specialized structure with enhanced sensitivity to fluid shear forces (aim 4).